Auswirkung der Nitrierung von Allergenen und von nitriertem Polytyrosin auf die Entwicklung einer akut allergischen Atemwegsreaktion am Mausmodell

Summary Within the last decades a rapid increase of allergic diseases such as bronchial asthma was reported, especially concerning the industrialized countries. The acute response of this chronic inflammatory disease with its hyperreactivity of the bronchial tract is characterized by activation of immunocompetent cells and their release of proinflammatory mediators, triggered by IgE. This process induces the clinical hallmarks of allergic asthma, such as acute airway obstruction, due to swelling of the bronchial mucosa, excessive secretion of mucus into the airways and contraction of smooth muscles. The late-phase response (2-24 h after the original reaction) is caused by activation of migrated leucocytes, especially eosinophils and an anew surge of inflammatory and bronchoconstrictive mediators. Large numbers of risk factors are discussed, as asthma is a multifactorial disease. Geographic distinctions of its incidence lead up to the conclusion there must be a correlation between the genetic background of a population and its environmental conditions, called „Gene-environmental-Interactions“. The present thesis deals with negative impacts on pulmonary health caused by traffic exhausts (keyword: Environmental factors). Various epidemiological and experimental studies revealed the damaging influence due to oxidative and nitrosative stress, in relation to aggravation or triggering of an allergic respiratory reaction. During the last years the formation of nitrotyrosine residues attached to proteins aroused special scientific attention, as this process is associated with numerous organic diseases. Exposure to oxidative and nitrosative species can decreases the redox potential of the airways severely. Subsequently a high rate of nitration in proteins/enzymes can induce dysfunctions. Patients suffering from inflammatory airway diseases showed to have a constantly elevated level of nitrotyrosine residues in their respiratory tract. Moreover it was demonstrated that high concentrations of ozone and nitrogen dioxide in congested areas effectively generate nitrated proteins/allergens that are inhaled and may exacerbate or trigger extrinsic-asthmatic reactions of the airways. For this study C57BL6- (n=228) and BALB/c-mice (n=54) were treated with nitrated and unmodified allergens (Ovalbumin (OVA, resp. nOVA) and keyhole limpet hemocyanin (KLH, resp. nKLH)) in a model of acute asthma. Three central hypotheses were postulated: 1. Nitration of allergens enhances their acute-inflammatory impact on the respiratory tract. 2. After sensitization against one specific nitrated allergen, other nitrated allergens induce similar allergic reactions in an individual, so that cross-reactions occur. 3. Treatment of BALB/c-mice with the nitrated amino acid polytyrosine arouses an allergic inflammatory reaction. After having the mice sensitized and provoked according to schedules for four weeks, various tests were performed to examine those hypotheses (timeframe: 2008 – 2011). Examinations on the bronchoalveolar lavage fluid (cell count, cell populations and concentration of proteins) and the histology showed to be most instructive. In particular the tested groups nOVA/nOVA, nKLH/nKLH and nOVA/nKLH, carried out with C57BL6- and BALB/c-mice, displayed cellular and structural changes due to inflammatory allergic reactions. Furthermore, type-II-pneumocytes were isolated from the lungs to extract proteins and RNA. Consequently central enzymes of the glutathione-system (glutathion reductase, glutathion peroxidase 3 and 4) were detected by immune-histochemical methods and by western blots using type-II-cell protein samples. In contrast to the controls, most of the tested groups showed an imposing increase in enzyme activity, exemplified in representative pictures. RT-qPCR gave an insight into DNA-expression of the named enzymes. As compared to the controls many of the tested groups displayed a reactive down-regulation on this level. All findings taken together, an aggravation of the allergic potential through nitration (first Hypothesis) was confirmed and examined in vivo for the first time. Treatment with nitrated allergens induced an intensified allergic inflammation of the airways, compared to treatment with unmodified allergens. The second Hypothesis (regarding cross reactions) was supported to some extent, yet more aimed investigations are required in future works. The third Hypothesis, involving nitrated polytyrosine, could not be verified by these exemplary tests on BALB/c-mice, as no characteristic signs of an allergic inflammation were detected.